The lysozyme from bacteriophage T4 will be used as a model system to understand the factors that determine the folding, stability, activity and three-dimensional structures of proteins. Specific research to be accomplished is as follows: 1. Mutant lysozymes with altered stability (and/or catalytic activity) will be selected and studied In detail. These mutant lysozymes will include not only temperature-sensitive lysozymes but also lysozymes that are more thermally stable than the wild-type enzyme. The three-dimensional structures of these mutant lysozymes will be determined and compared with the wild-type lysozyme structure. Changes in structure will be correlated with changes in thermodynamics and kinetics of folding. 2. Oligonucleotide-directed mutagenesis will be used to introduce designed alterations in the lysozyme structure. By making one or more selected amino acid replacements at a given site we will discriminate between the contributions of different Interactions at that site. Site-directed and saturation mutagenesis will also be used to test the importance of factors such as hydrogen bonding, secondary structure stabilization, hydrophobic interaction, salt bridges, van der Waals interactions, disulfide bridges, metal binding sites, and stearic hindrance in protein folding and stability. 3. An attempt will be made to simplify the protein folding problem by identifying which residues in T4 lysozyme are critical for folding and stability, and which are not. Ultimately we would like to reduce the amino acid sequence of T4 lysozyme to the simplest form that will still give a folded, functional protein.